Vaccines with enhanced immune response and methods for their preparation

ABSTRACT

The present invention is concerned with vaccines and their preparation. An effective long-term immune response, especially in mammals, can be produced using a vaccine comprising an antigen encapsulated in liposomes, a suitable adjuvant and a carrier comprising a continuous phase of a hydrophobic substance. The vaccine is particularly effective in eliciting the production of antibodies that recognize epitopes of native proteins.

RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplications Ser. No. 60/246,075 filed Nov. 7, 2000 and U.S. Ser. No.60/307,159 filed Jul. 24, 2001, the disclosures of which are herebyincorporated by reference in their entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of immunology, inparticular, to vaccines and their preparation.

BACKGROUND OF THE INVENTION

[0003] Generally, vaccines use low doses of a specific antigen to buildup resistance in a host to the effects of larger doses of the antigen orsimilar antigenic compounds. Antigens used in vaccines are usually partsof whole organisms or denatured toxins (toxoids) that induce theproduction of antibodies. Unfortunately, only some of the antibodiesproduced bind to the target organism or toxin because, in most cases,the antigen used in the vaccine differs structurally from the target.The limited availability of useful antigens has posed limitations tovaccine development in the past. Advances in genetic engineering havemade the production of antigens by recombinant means possible. However,use of antigens produced by recombinant means often results in poorproduction of antibodies with poor affinity for the target nativeantigen for reasons given above. The effect of immunization can beenhanced when more antibodies with high affinity for their target areproduced. There is a need in the art to develop vaccines that produce anenhanced immune response without increasing the amount of antigen usedin the vaccine. Particularly, there is a need for single administrationvaccines that eliminate or reduce the need for booster immunizations.

[0004] Many immunization strategies would benefit from such development.Vaccines that use antigens derived from mammalian, viral, bacterial,fungal or yeast sources have many uses. For example, antigens fromviral, bacterial, fungal or yeast sources are useful in the preventionof disease. Antigens from mammals may be used in cancer therapy orimmunocontraception. Immunocontraceptive vaccines use mammalian derivedantigens that result in transient infertility or sterility of a host,particularly a mammalian host, by favouring the production of antibodieswith affinity for the oocyte surface. Immunocontraceptive vaccines finduse in the control of wild animal populations, including populations offeral domestic animals such as cats.

[0005] In particular, feral cat populations have been difficult tocontrol and threaten many birds and small animals. Stray feral cats alsoact as vectors for human and animal diseases. Various methods includinghunting, trapping and poisoning have been used in an effort to controlstray cat populations but these methods have met with limited successand with public opposition. Surgical sterilization of feral cats hasbeen increasingly used as a humane tool to lower feral cat populationsduring the last two decades. Acceptance of this procedure is widespread;however, disadvantages include cost, changes in behaviour and risk ofinfection and mortality. Despite the success of large-scale surgicalsterilization, such programs are not financially or logisticallyfeasible in many locations since capture of animals is time-consuming,difficult and stressful for the animal. Immunocontraception offers analternate procedure with lower costs and ease of administration.However, long-term immunocontraception generally requires boostervaccinations, making it impractical for the control of wild andfree-roaming species.

[0006] Vaccines generally comprise an antigen, which elicits the immuneresponse in the host, and a variety of carriers, excipients andadjuvants useful for administering the antigen to the host.

[0007] Liposomes, which encapsulate the antigen, have increasingly beenused in vaccine delivery. It has been shown that liposome delivery ofdenatured antigens favours the production of antibodies that recognizenative epitopes (Muttilainen, S., I. Idanpaan-Heikkila, E. Wahlstrom, M.Nurminen, P. H. Makela and M. Sarvas. 1995. “The Neisseria meningitidisouter membrane protein P1 produced in Bacillus subtilis andreconstituted into phospholipid vesicles elicits antibodies to native P1epitopes.” Microbial Pathogen. 18:423-436). While liposomes are usefulvaccine delivery vehicles, their use alone has not provided an effectivesingle dose vaccine, particularly with respect to immunocontraceptivevaccines.

[0008] Most immunocontraceptive vaccines use Freund's Complete Adjuvant(FCA) followed by Freund's Incomplete Adjuvant (FIA) in multipleinjections to aid production of sufficient antibodies to have animmunocontraceptive effect (see Ivanova, et al., 1995. “Contraceptivepotential of porcine zona pellucida in cats.” Theriogenology. 43:969-981and Sacco et al., 1989. “Effect of varying dosage and adjuvants onantibody response in squirrel monkeys (Saimiri sciureus) immunized withthe porcine zona pellucida Mr=55,000 glycoprotein (ZP3).” Am. J. Reprod.Immunol. 21:1-8). Other adjuvants such as Ribi™ and TiterMax™ have beenused by some investigators. Alum (aluminum phosphate and/or hydroxide)has a long history of use as an adjuvant. Alum is the only adjuvantrecognized as safe by the Food and Drug Administration. Manyimmunocontraceptive vaccines that use alum require a primary injectionand several booster injections to produce sufficient antibodies for animmunocontraceptive effect (see Bagavant et al., 1994. “Antifertilityeffects of porcine zona pellucida-3 immunization using permissibleadjuvants in female bonnet monkeys (Macaca radiata): reversibility,effect on follicular development and hormonal profiles.” J. Reprod.Fertil. 102:17-25). Some studies have shown that alum is not a suitableadjuvant for zona pellucida immunocontraceptive vaccines (see Sacco etal., 1989. Am. J. Reprod. Immunol. 21:1-8 and Bagavant et al., 1994. J.Reprod. Fertil. 102:17-25).

[0009] Prior art has generally relied on the use of an aqueous medium oroil-in-water emulsions as carriers. For example, Muttilainen et al.(Microbial Pathogen. 18:423-436 (1995) use an aqueous medium incombination with liposomal delivery to elicit an immune response.Popescu (U.S. Pat. No. 5,897,873 issued Apr. 27, 1999 and U.S. Pat. No.6,090,406 issued Jul. 18, 2000), Alving (U.S. Pat. No. 6,093,406 issuedJul. 25, 2000 and U.S. Pat. No. 6,110,492 issued Aug. 29, 2000) andMuderhwa et al. (“Oil-in-water liposomal emulsions: Characterization andpotential use in vaccine delivery”, (December, 1999) J Pharm Sci.88(12):1332-9) also use liposomal systems together with an oil-in-watercarrier as the delivery system in a vaccine. Popescu uses alum withliposomes consisting of cholesterol esterified with succinate or otherorganic acids. U.S. Pat. No. 6,093,406 teaches the use of alum andliposomes comprising Lipid A or non-pyrogenic Lipid A in an oil-in-wateremulsion to deliver a vaccine based on malarial antigens. U.S. Pat. No.6,110,492 and Muderhwa teach the use of liposomes comprising Lipid A ornon-pyrogenic Lipid A in an oil-in-water emulsion to deliver prostratespecific antigens.

[0010] Commonly owned U.S. Pat. No. 5,736,141, issued on Apr. 7, 1998,teaches a single dose immunocontraceptive vaccine for seals derived fromzona pellucida antigens. While the results achieved with this vaccineare good, there is still a need for a single-dose, long lastingimmunocontraceptive vaccine effective in a variety of species usingadjuvants approved by the Food and Drug Administration.

[0011] There also remains a need for long lasting immunovaccines ingeneral which are effective using a variety of antigens in a variety ofspecies using adjuvants approved by the Food and Drug Administration.

SUMMARY OF THE INVENTION

[0012] In accordance with the invention, there is provided a compositionfor use as a vaccine, comprising:

[0013] (a) a carrier comprising a continuous phase of a hydrophobicsubstance;

[0014] (b) liposomes;

[0015] (c) an antigen; and,

[0016] (d) a suitable adjuvant.

[0017] There is further provided a method for potentiating an immuneresponse in an animal, which method comprises administering to theanimal an effective amount of a vaccine composition comprising:

[0018] (a) a carrier comprising a continuous phase of a hydrophobicsubstance;

[0019] (b) liposomes;

[0020] (c) an antigen; and,

[0021] (d) a suitable adjuvant.

[0022] Still further there is provided a method of preparing a vaccinecomposition comprising the steps of:

[0023] (a) encapsulating an antigen or an antigen/adjuvant complex inliposomes to form liposome-encapsulated antigen;

[0024] (b) mixing the liposome-encapsulated antigen with a carriercomprising a continuous phase of a hydrophobic substance; and,

[0025] (c) adding a suitable adjuvant if an antigen/adjuvant complex isnot used in part (a).

[0026] Unexpectedly and uniquely, it has now been found that using acontinuous phase of a hydrophobic substance as the carrier in a vaccinecomposition of the present invention enhances the immune response. Theenhanced response is characterized by long-lived high antibody titresfollowing a single vaccine administration resulting in enhanced durationof the immune response. This is particularly true for vaccines that alsocomprise liposome-encapsulated antigen and an adjuvant (or mixture ofantigen/adjuvant). Vaccine compositions of the present invention aregenerally effective as a single dose providing a long-term immuneresponse in a variety of species, typically not requiring boosters.

DETAILED DESCRIPTION OF THE INVENTION

[0027] While not being held to any particular theory of action, it isthought that, when a vaccine composition of the present invention isused, IgG antibody production occurs in two phases and the antibodiesproduced in each phase differ in their epitope recognition. Theantibodies produced in the second phase of IgG production have moreaffinity for native protein antigens, thus making the vaccine moreeffective. Use of conventional vaccines with a primary and boosterinjection produces antibodies having different binding specificity foran antigen than use of a vaccine composition of the present invention.

[0028] The carrier comprises a continuous phase of a hydrophobicsubstance, preferably a liquid hydrophobic substance. The continuousphase may be an essentially pure hydrophobic substance, a mixture ofhydrophobic substances, an emulsion of water-in-a hydrophobic substanceor an emulsion of water-in-a mixture of hydrophobic substances.

[0029] Hydrophobic substances that are useful in the present inventionare those that are pharmaceutically and/or immunologically acceptable.Ideally, the hydrophobic substance is one that has been approved for useby health regulatory agencies such as the U.S. Food and DrugAdministration. The carrier is preferably a liquid but certainhydrophobic substances that are not liquids at atmospheric temperaturemay be liquified, for example by warming, and are also useful in thisinvention.

[0030] Oil or water-in-oil emulsions are particularly suitable carriersfor use in the present invention. Oils should be pharmaceutically and/orimmunologically acceptable. Preferred examples of oils are mineral oil(especially light or low viscosity mineral oil), vegetable oil (e.g.corn or canola oil), nut oil (e.g. peanut oil) and squalene. A lowviscosity mineral oil is most preferred. Animal fats and artificialhydrophobic polymeric materials, particularly those that are liquid atatmospheric temperature or that can be liquified relatively easily, mayalso be used.

[0031] The amount of hydrophobic substance used is not critical but istypically from about 0.1 ml per dose to about 1.5 ml per dose, dependingon the size of the animal and the amount of antigen being used. Forsmall animals, the amount of hydrophobic substance is preferably fromabout 0.20 ml to about 1.0 ml per dose, while for large animals, theamount is preferably from about 0.45 ml to about 1.5 ml per dose.Typically, 0.25 ml per dose is used for small animals while 0.5 ml perdose is used for large animals.

[0032] Suitable antigens are any chemicals that are capable of producingan immune response in a host organism. Preferably, the antigen is asuitable native, non-native, recombinant or denatured protein orpeptide, or a fragment thereof, that is capable of producing the desiredimmune response in a host organism. Host organisms are preferablyanimals (including mammals), more preferably cats, rabbits, horsesand/or deer. The antigen can be of a viral, bacterial, protozoal ormammalian origin. Antigens are generally known to be any chemicals(typically proteins or other peptides) that are capable of eliciting animmune response in a host organism. More particularly, when an antigenis introduced into a host organism, it binds to an antibody on B cellscausing the host to produce more of the antibody. For a generaldiscussion of antigens and the immune response, see Kuby, J., Immunology3^(rd) Ed. W.H. Freeman & C. NY (1997), the disclosure of which ishereby incorporated by reference.

[0033] Antigens that elicit an immune response related to cancer,contraception and other biological conditions or effects may be used inthe preparation of immunovaccines. Some typical, non-limiting examplesof antigens that may be used are alcohol dehydrogenase (ADH),streptokinase, hepatitis B surface antigen and zona pellucida (ZP)glycoproteins.

[0034] When the desired immune response is contraception in mammals, thetarget epitopes are found on mammalian oocytes. Zona pellucida (ZP)glycoproteins or recombinant proteins or peptide fragments derivedtherefrom may be used in this case. In particular, heat extractedsolubilized isolated zona pellucida glycoproteins (SIZP) may be used asthe antigen in an immunocontraceptive vaccine. More particularly,soluble intact porcine zona pellucida may be used.

[0035] The amount of antigen used in a dose of the vaccine compositioncan vary depending on the type of antigen and the size of the host. Oneskilled in the art will be able to determine, without undueexperimentation, the effective amount of antigen to use in a particularapplication.

[0036] In the case of SIZP, the amount typically used falls in the rangefrom about 15 μg to about 2 mg per dose. Preferably, the range is fromabout 20 μg to about 2 mg per dose, more preferably from about 20 μg toabout 200 μg, and even more preferably from about 40 μg to about 120 μg.Typically, the amount for a small animal is about 50 μg per dose whilefor a large animal it is about 100 μg per dose.

[0037] In compositions of the present invention, antigens produceenhanced levels of host antibodies that bind to native epitopes of thetarget protein. This is the case even though the antigen may be anon-native, recombinant or denatured protein or peptide, or a fragmentthereof. While not wishing to be held to any particular theory, this maybe due to the antigen being held in a native-like three-dimensionalconformation in the liposomes.

[0038] Liposomes are completely closed lipid bilayer membranescontaining an entrapped aqueous volume. Liposomes may be unilamellarvesicles (possessing a single bilayer membrane) or multilamellarvesicles (onion-like structures characterized by multimembrane bilayers,each separated from the next by an aqueous layer. A general discussionof liposomes can be found in Gregoriadis G.(1990) Immunologicaladjuvants: A role for liposomes, Immunol. Today 11:89-97 and Frezard, F.(1999) Liposomes: From biophysics to the design of peptide vaccines.Braz. J. Med. Bio. Res 32:181-189, the disclosures of which are herebyincorporated by reference.

[0039] Although any liposomes may be used in this invention, includingliposomes made from archaebacterial lipids, particularly usefulliposomes use phospholipids and unesterified cholesterol in the liposomeformulation. The cholesterol is used to stabilize the liposomes and anyother compound that stabilizes liposomes may replace the cholesterol.Other liposome stabilizing compounds are known to those skilled in theart. The use of the particularly preferred liposomes may result inlimiting the electrostatic association between the antigen and theliposomes. Consequently, most of the antigen may be sequestered in theinterior of the liposomes.

[0040] Phospholipids that are preferably used in the preparation ofliposomes are those with at least one head group selected from the groupconsisting of phosphoglycerol, phosphoethanolamine, phosphoserine,phosphocholine and phosphoinositol. More preferred are liposomes thatcomprise lipids in phospholipon 90 G.

[0041] The amount of lipid used to form liposomes depends on the antigenbeing used but is typically in a range from about 0.05 gram to about 0.5gram per dose of vaccine. Preferably, the amount is about 0.1 gram perdose. When unesterified cholesterol is also used in liposomeformulation, the cholesterol is used in an amount equivalent to about10% of the amount of lipid. The preferred amount of cholesterol is about0.01 gram per dose of vaccine. If a compound other than cholesterol isused to stabilize the liposomes, one skilled in the art can readilydetermine the amount needed in the formulation.

[0042] In a more preferred aspect, the vaccine compositions of thepresent invention are essentially free from Lipid A, includingnon-pyrogenic Lipid A. For the purposes of this specification, when theterm Lipid A is used, it is understood to encompass non-pyrogenic LipidA as well. Lipid A is often found in liposomal formulations of the priorart. Lipid A has many undesirable side-effects which may be overcomeusing non-pyrogenic Lipid A, but even then, Lipid A has manypharmaceutical reactions other than the pyrogenic one and may stillcause many adverse reactions. It is therefore desirable to exclude LipidA from the compositions of this invention.

[0043] Suitable adjuvants are alum, other compounds of aluminum,Bacillus of Calmette and Guerin (BCG), TiterMax™, Ribi™, Freund'sComplete Adjuvant (FCA) and a new adjuvant disclosed by the UnitedStates Department of Agriculture's (USDA) National Wildlife ResearchCenter on their web site at http://www.aphis.usda.gov/ws/nwrc/pzp.htmbased on Johne's antigen. Alum, other compounds of aluminum, TiterMax™and the new USDA adjuvant are preferred. Enhanced immune response isfound even when the adjuvant is alum, which is surprising in view of theprior art (Sacco et al. 1989. Am. J. Reprod. Immunol., 21:1-8). Alum isparticularly preferred as the adjuvant.

[0044] Alum is generally considered to be any salt of aluminum, inparticular, the salts of inorganic acids. Hydroxide and phosphate saltsare particularly useful as adjuvants. A suitable alum adjuvant is soldunder the trade name, ImjectAlum™ (Pierce Chemical Company) thatconsists of an aqueous solution of aluminum hydroxide (45 mg/ml) andmagnesium hydroxide (40 mg/ml) plus inactive stabilizers. Alum is aparticularly advantageous adjuvant since it already has regulatoryapproval and it is widely accepted in the art.

[0045] The amount of adjuvant used depends on the amount of antigen andon the type of adjuvant. One skilled in the art can readily determinethe amount of adjuvant needed in a particular application. Forimmunocontraception, a suitable quantity of ImjectAlum™ for a rabbit is0.1 ml/dose of vaccine, whereas, a suitable quantity of ImjectAlum™ fora horse is 0.5 ml/dose.

[0046] The vaccine composition is generally formulated by: encapsulatingan antigen or an antigen/adjuvant complex in liposomes to formliposome-encapsulated antigen and mixing the liposome-encapsulatedantigen with a carrier comprising a continuous phase of a hydrophobicsubstance. If an antigen/adjuvant complex is not used in the first step,a suitable adjuvant may be added to the liposome-encapsulated antigen,to the mixture of liposome-encapsulated antigen and carrier, or to thecarrier before the carrier is mixed with the liposome-encapsulatedantigen. The order of the process may depend on the type of adjuvantused. Typically, when an adjuvant like alum is used, the adjuvant andthe antigen are mixed first to form an antigen/adjuvant complex followedby encapsulation of the antigen/adjuvant complex with liposomes. Theresulting liposome-encapsulated antigen is then mixed with the carrier.(It should be noted that the term “liposome-encapsulated antigen” mayrefer to encapsulation of the antigen alone or to the encapsulation ofthe antigen/adjuvant complex depending on the context.) This promotesintimate contact between the adjuvant and the antigen and may, at leastin part, account for the surprisingly good immune response when alum isused as the adjuvant. When another is used, the antigen may be firstencapsulated in liposomes and the resulting liposome-encapsulatedantigen is then mixed into the adjuvant in a hydrophobic substance.

[0047] In formulating a vaccine composition that is substantially freeof water, antigen or antigen/adjuvant complex is encapsulated withliposomes and mixed with a hydrophobic substance. In formulating avaccine in an emulsion of water-in-a hydrophobic substance, the antigenor antigen/adjuvant complex is encapsulated with liposomes in an aqueousmedium followed by the mixing of the aqueous medium with a hydrophobicsubstance. In the case of the emulsion, to maintain the hydrophobicsubstance in the continuous phase, the aqueous medium containing theliposomes may be added in aliquots with mixing to the hydrophobicsubstance.

[0048] In all methods of formulation, the liposome-encapsulated antigenmay be freeze-dried before being mixed with the hydrophobic substance orwith the aqueous medium as the case may be. In some instances, anantigen/adjuvant complex may be encapsulated by liposomes followed byfreeze-drying. In other instances, the antigen may be encapsulated byliposomes followed by the addition of adjuvant then freeze-drying toform a freeze-dried liposome-encapsulated antigen with externaladjuvant. In yet another instance, the antigen may be encapsulated byliposomes followed by freeze-drying before the addition of adjuvant.Freeze-drying may promote better interaction between the adjuvant andthe antigen resulting in a more efficacious vaccine.

[0049] Formulation of the liposome-encapsulated antigen into ahydrophobic substance may also involve the use of an emulsifier topromote more even distribution of the liposomes in the hydrophobicsubstance. Typical emulsifiers are well-known in the art and includemannide oleate (Arlacel™ A), lecithin, Tween™ 80, Spans™ 20,80, 83 and85. Mannide oleate is a preferred emulsifier. The emulsifier is used inan amount effective to promote even distribution of the liposomes.Typically, the volume ratio (v/v) of hydrophobic substance to emulsifieris in the range of about 5:1 to about 15:1 with a ratio of about 10:1being preferred.

[0050] Administration of the vaccine composition can be done by anyconvenient method and will depend on the antigen being used. Vaccinecompositions may be administered parenterally (includingintramuscularly, sub-cutaneously) or rectally. Parenteral administrationis preferred.

[0051] For parenteral application, particularly convenient unit dosageforms are ampoules. Techniques that deliver the vaccine by injection andby remote delivery using darts, spring loaded syringes with jab sticks,air/carbon dioxide powered rifles, Wester gun and/or Ballistive™biobullets and retain the biological activity are particularlypreferred.

[0052] The amount of vaccine composition administered to a host maydepend on the amount of antigen used in a dose and on the effectiveamount of antigen required for a particular application. In the case ofSIZP, the size of each dose administered to an animal is typically fromabout 0.25 ml to about 2.0 ml depending on the size of the animal. Forsmaller animals (for example, cats, rabbits, etc.) the size of the doseis typically about 0.5 ml while for larger animals (for example, horses,fallow-deer, white-tail deer, etc.) the size of the dose is typicallyabout 1.0 ml. Typically, even when the amount of SIZP is varied, thedose size is kept fairly constant.

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The invention will now be described by way of non-limitingexamples having regard to the appended drawing in which:

[0054]FIG. 1 is a diagram showing the position of recombinant ZPB1 andZPB2; ZPC1 and ZPC2 of ZPB and ZPC proteins of porcine zona pellucidathat were generated in the PRSET vectors.

EXAMPLES Example 1 Preparation of the Vaccine Composition

[0055] The vaccine composition can be formulated to be water-free or tocontain various quantities of water (by using an aqueous medium, forexample, saline, phosphate buffered saline (PBS) or pyrogen-free water)while maintaining a continuous oil phase. Procedure 1 described belowapplies to the water-free formulation of the vaccine composition.Procedure 2 described below applies to the water containing formulationof the vaccine composition, that is, the water-in-oil emulsion. The twoprocedures can also vary depending on the adjuvant being used. Asexamples, method A applies to formulations of the vaccine compositioncontaining alum and method B applies to formulations containing Freund'sComplete Adjuvant (FCA). Other adjuvants may be accommodated by adaptingeither method A or method B. The procedures described below incorporateporcine soluble intact zona pellucida (SIZP) as antigen, other antigenscan replace SIZP in the formulation. For example, alcohol dehydrogenase(ADH), streptokinase or hepatitis B surface antigen can also be used asthe antigen.

[0056] Procedure 1: Water-free Formulation.

[0057] Method A. Alum Adjuvant.

[0058] SIZP is prepared as previously described (Brown, R. G., W. D.Bowen, J. D. Eddington, W. C. Kimmins, M. Mezei, J. L. Parsons, B.Pohajdak. (1997) Temporal trends in antibody production in captive greyseals, harp and hooded seals to a single administrationimmunocontraceptive vaccine. J. Reproductive Immunology 35:53-64). Thequantity of SIZP needed for the number of doses of the vaccine beingprepared is weighed (the usual quantity of SIZP used for immunization is50 μg for small animals and 100 μg for large animals). The SIZP isdissolved in pyrogen-free distilled water to give a final concentrationof 2 mg/ml. An equal volume of ImjectAlum™ (an alum product from PierceChemical Co., catalogue # 77161) is added and the suspension is mixed,then freeze-dried.

[0059] To form liposomes, phospholipon 90 G (or other lipids selectedfrom phosphoglycerol, phosphoethanolamine, phosphoserine,phosphocholine, phosphoinositol, archaebacterial lipids, withoutlimitation, that form a closed lipid bilayer containing an entrappedantigen) is weighed (0.1 g/dose of the vaccine composition). Thephospholipon 90 G is mixed with cholesterol (0.01 g/dose of vaccinecomposition) and the mixture is dissolved in chloroform:methanol(1/1;v/v; 1.5 ml/dose of the vaccine composition). Cholesterol can bereplaced with other compounds that stabilize liposomes at concentrationsdetermined by those skilled in the art. Washed glass beads(approximately 3 mm in diameter; 15 ml for 10 doses of the vaccine) areadded and the mixture is evaporated under reduced pressure using arotary evaporator until free of chloroform:methanol. To ensure removalof all chloroform:methanol, the mixture is placed in a dessicator underreduced pressure overnight at room temperature.

[0060] The freeze-dried SIZP/alum complex is suspended in pyrogen-freedistilled water (5 ml/mg SIZP) and the suspension added to the flaskcontaining the mixture of phospholipon 90 G/cholesterol coating theflask and glass beads. The contents of the flask are allowed to standwithout agitation for 30 minutes. After 30 minutes, the flask is placedin a water bath at 35-40° C. and stirred gently with a spatula to formthe liposomes. A microscope is used to evaluate liposome formation andstirring is continued with increased shaking until the mixture containspredominately multilamellar liposomes recognized by those skilled in theart. The liposomes are freeze-dried and the resulting freeze-driedliposomes are suspended in low viscosity mineral oil (0.25 ml oil/dosefor small animals and 0.5 ml oil/dose for large animals) containingmannide oleate as an emulsifier (10:1:oil:emulsifier:v/v). Sinceliposomes are suspended in oil and are not in solution, it is necessaryto determine if the procedures used result in an even distribution ofSIZP in each dose. To determine if freeze-dried liposomes containingSIZP are equally distributed in oil, SIZP is labelled with ¹⁴C byreductive methylation (Jentoft, N. and D. G. Dearborn. 1979. Labellingof proteins by reductive methylation using sodium cyanoborohydride. J.Biol. Chem. 254:4359-4365, the disclosure of which is herebyincorporated by reference) and the radioactive SIZP used to prepare twopreparations of the vaccine. Individual doses of the vaccine areprepared and radioactivity in each dose determined, thereby determiningthe content of SIZP in each dose of the vaccine (Table 1). Thedistribution of SIZP in each dose of the vaccine is highly reproducible(standard deviation, SD, was less than +/−10%). TABLE 1 Distribution of¹⁴C-labelled SIZP in doses of the vaccine from two preparations SamplePreparation 1 Preparation 2 No. μg SIZP/dose μg SIZP/dose 1 68 55 2 6653 3 57 53 4 65 62 5 56 59 6 51 57 7 62 58 8 55 60 9 69 51 10 54 59 1152 57 12 61 61 13 64 61 14 61 60 15 — 56 Average 60 57 Standard 5.9 3.2Deviation

[0061] Preparation of the vaccine composition to contain FCA as adjuvantin place of alum, is similar to method A, except SIZP by itself, ratherthan as a SIZP/alum complex, is encapsulated in liposomes as describedabove. The liposomes containing SIZP are freeze-dried, and thefreeze-dried liposomes are added to FCA in aliquots with mixing topromote an equal distribution of liposomes in the oil. The resultingsuspension of freeze-dried liposomes containing SIZP in FCA isadministered to animals being vaccinated.

[0062] Procedure 2. Water-Containing Formulation.

[0063] Method A. Alum Adjuvant.

[0064] SIZP is prepared as previously described (Brown, R. G., W. D.Bowen, J. D. Eddington, W. C. Kimmins, M. Mezei, J. L. Parsons, B.Pohajdak. (1997) Temporal trends in antibody production in captive greyseals, harp and hooded seals to a single administrationimmunocontraceptive vaccine. J. Reproductive Immunology 35:53-64, thedisclosure of which is hereby incorporated by reference). The quantityof SIZP needed for the number of doses of the vaccine being prepared isweighed (the usual quantity of SIZP used for immunization is 50 μg forsmall animals and 100 μg for large animals). The SIZP is dissolved inpyrogen-free distilled water to give a final concentration of 2 mg/ml.An equal volume of ImjectAlum™ (an alum product from Pierce ChemicalCo., catalogue # 77161) is added and the suspension is mixed, thenfreeze-dried.

[0065] To form liposomes, phospholipon 90 G (or other lipids selectedfrom phosphoglycerol, phosphoethanolamine, phosphoserine,phosphocholine, phosphoinositol, etc. that form a closed lipid bilayercontaining an entrapped aqueous volume) is weighed (0.1 g/dose of thevaccine composition). The phospholipon 90 G is mixed with cholesterol(0.01 g/dose of the vaccine composition) and the mixture is dissolved inchloroform:methanol (1/1;v/v; 1.5 ml/dose of the vaccine composition).Cholesterol can be replaced with other compounds that stabilizeliposomes at concentrations determined by those skilled in the art.Washed glass beads (approximately 3 mm in diameter; 15 ml for 10 dosesof the vaccine) are added and the mixture is evaporated under reducedpressure using a rotary evaporator until free of chloroform:methanol. Toensure removal of all chloroform:methanol, the mixture is placed in adessicator under reduced pressure overnight at room temperature.

[0066] The freeze-dried SIZP/alum complex is suspended in saline (5ml/mg SIZP) and the suspension is added to the flask containing themixture of phospholipon 90 G/cholesterol coating the flask and glassbeads. The contents of the flask are allowed to stand without agitationfor 30 minutes. After 30 minutes, the flask is placed in a water bath at35-40° C. and stirred gently with a spatula to form the liposomes. Amicroscope is used to evaluate liposome formation and stirring iscontinued with increased shaking until the mixture containspredominately multilamellar liposomes recognized by those skilled in theart. The aqueous suspension of liposomes (0.25 ml/dose for small animalsand 0.5 ml/dose for large animals) is added to low viscosity mineral oil(0.25 ml oil/dose for small animals and 0.5 ml oil/dose for largeanimals) containing mannide oleate as an emulsifier(10:1:oil:emulsifier:v/v). The aqueous suspension of liposomes is addedto the low mineral oil phase in aliquots with mixing between aliquots tomaintain the continuous oil phase.

[0067] Method B. FCA Adjuvant.

[0068] Preparation of the vaccine composition to contain FCA as adjuvantin place of alum, is similar to method A, except SIZP by itself, ratherthan as a SIZP/alum complex, is encapsulated in liposomes as describedabove. The aqueous suspension of liposomes is added to FCA in aliquotswith mixing between aliquots to maintain the continuous oil phase. Theresulting aqueous suspension of liposomes containing SIZP in FCA isadministered to animals being vaccinated.

[0069] Note: In some trials, the quantity of SIZP is varied to study theresponse of immunized animals to different quantities of antigen. Insuch experiments, the volume of the vaccine administered to smallanimals (cats, rabbits, etc.) was 0.5 ml and the volume administered tolarge animals (horses, fallow deer, white-tailed deer, etc.) was 1.0 ml.In such cases, the quantity of liposomes in each dose of the vaccine ismaintained constant while the quantity of antigen encapsulated inliposomes varied.

Example 2 Immunization of Rabbits Against Native and Denatured YeastAlcohol Dehydrogenase (ADH)

[0070] The vaccine composition is unique in producing high titers ofanti-SIZP antibodies that are long-lasting following a singleadministration. To determine if the vaccine composition would producehigh antibody titers with other antigens, particularly proteins that arenot bound to cell membranes, rabbits are immunized with yeast alcoholdehydrogenase (ADH). Two forms of ADH are used as antigen, namely,native ADH and ADH that had been treated to denature the protein. Todenature ADH, ADH is treated with mercaptoethanol (10% v/v in Trisbuffer, 0.1 M, pH 7.5, 30 min, 100° C.). The solution is dialyzedagainst distilled water and freeze-dried. Four rabbits (2 for eachtreatment) are immunized with native or denatured ADH (40 μg) using aprimary injection with Freund's complete adjuvant (FCA) followed by abooster injection with Freund's incomplete adjuvant (FIA) given onemonth later. The post-immunization period is considered to have begunafter the booster injection. At the same time as the booster injectionis administered, four rabbits (2 for each treatment) are immunized bysingle administration with either native or denatured ADH (40 μg) usingthe vaccine composition, that is ADH is encapsulated in liposomes thatare suspended in saline (0.5 ml) and emulsified in FCA (0.5 ml).Anti-ADH titers are measured by ELISA using both native and denaturedADH (Table 2).

[0071] When rabbits are immunized with native ADH, the resulting serumcontained similar quantities of anti-ADH antibodies when native ADH isdelivered with the vaccine composition or by using a primary injectionwith one booster. In contrast, serum from rabbits immunized withdenatured ADH delivered with the vaccine composition contain 2.7 timesmore antibody that bound to native ADH than serum from rabbits that areimmunized with denatured ADH with a primary injection and one booster(P<0.01; T=4.14; df=6). In all cases, titers are higher in rabbitsimmunized with native ADH than when rabbits were immunized withdenatured ADH. This indicates that native ADH is a better antigen thandenatured ADH. Since many protein antigens are denatured to some degreeduring extraction and isolation or when produced by recombinant means,increased production of antibodies that bind better to native proteinscan significantly improve the outcome of vaccination as demonstrated byimmunocontraception of a variety of mammals with SIZP using the vaccinecomposition of the present invention.

[0072] Furthermore, anti-ADH sera from rabbits 237 and 238 recognizedenatured ADH in Western blots with about 4-5 times the intensity ofanti-ADH sera from rabbits 235 and 236. This confirms the results oftiter measurements indicating that immunization of rabbits with thevaccine composition favours the production of anti-ADH antibodies thatbind better to native ADH since many proteins are known to refold duringthe Western protocol to a more native state. This conclusion issupported by Muttilainen et al. (1995) in a study of Neisseriameningitidis outer membrane protein Pi, who found antibodies to nativeP1 were elicited in mice vaccinated with denatured P1 constituted intophospholipid vesicles (liposomes). However, Muttilainen et al. (1995)did not use oil in their vaccine formulation, therefore, theirimmunization protocol was different than the present invention. TABLE 2Production of anti-ADH antibodies by rabbits immunized with native ordenatured ADH delivered with and without liposome encapsulation Anti-ADHtiter (% of reference serum)¹ Immunization Denatured Rabbit Native ADH³ADH³ ID No. Antigen Delivery² 4⁴ 5⁴ 4⁴ 5⁴ 231 native −Liposomes 187 14819 23 ADH 232 native −Liposomes 200 161 17 15 ADH 233 native +Liposomes239 156 21 14 ADH 234 native +Liposomes 100 100 19 11 ADH 235 denatured−Liposomes  41  37  7  3 ADH 236 denatured −Liposomes  30  18  7  4 ADH237 denatured +Liposomes 101  71  8  3 ADH 238 denatured +Liposomes 101 63 12  7 ADH

Example 3 Immunocontraception of Rabbits

[0073] Sera from rabbits immunized with a placebo vaccine that containedall ingredients of the vaccine composition except the antigen (porcineSIZP) contain no anti-porcine SIZP antibodies (see Table 3A).Immunization of rabbits with porcine SIZP (40 μg) encapsulated inliposomes containing phosholipon 90 G (0.1 g), cholesterol (0.01 g) insaline (0.5 ml) emulsified in FCA adjuvant (0.5 ml) produce high titersof anti-SIZP antibodies during the 12 month post-immunization monitoringperiod following a single administration of the vaccine. Immunization ofrabbits with porcine SIZP (40 μg) encapsulated in liposomes with MF 59adjuvant (0.5 ml) produce low anti-SIZP titers. In contrast,immunization of rabbits with porcine SIZP encapsulated in liposomes withalum adjuvant (100 μl, Pierce ImjectAlum™) produce anti-porcine SIZPtiters that are less than titers produced using FCA in earlypost-immunization but the titers are less different than between thealum and FCA runs by the 12^(th) month of post-immunization. Breeding ofrabbits established that a single administration of the vaccine usingFCA or alum reduces fertility of rabbits by 79 and 74% respectively(Table 3B). Immunization of rabbits by a single injection of SIZP (40μg) that is not encapsulated in liposomes with Gerbu adjuvant produceslow anti-porcine SIZP titers (Table 3A). As expected based on anti-SIZPtiters, rabbits immunized with SIZP that are not encapsulated inliposomes with Gerbu adjuvant have the same fertility as rabbits thatreceive the placebo vaccine (Table 3B). These results indicate thatvaccines comprising liposome-encapsulated antigen produce good resultsand that FCA and alum, particularly alum, are especially good adjuvants.TABLE 3A Effect of adjuvants on the production of anti-porcine SIZPantibodies by rabbits¹ Post-immunization anti-porcine SIZP titer (% ofreference ID Time (months) No. 0 1 2 3 4 5 6 7 11 12 Placebo  2 0  1  0 0  0  0  0  0  0  0 13 0  0  0  0  0  0  0  0  0  0 14 0  0  0  0  0  0 0  0  0  0 FCA  3 0 145 112  94  82 45 23 24 20 20 15 0 100  71  68  83 19 29 26  18 25 16 0 125 111 122 128 111 74 92 75 63 MF 59  1 0  3  1 1  1  2  0 ND ND ND  7 0  10  9  3  3  5  2 ND ND ND 19 0  12  6  3  3 3  3 ND ND ND 20 0  37  21  13  15  13 10 ND ND ND Alum 11 0  19  12 12  11  14 18  7  8 10 12 0  20  10  10  8  6 11  5  2  4 23 0  31  16 40  22  20 33 28  37 28 24 0  36  21  40  27  24 40 36  34 34 Gerbuwithout liposome encapsulation of SIZP  9 0  5  2  1  1  1  1  1 ND ND10 0  11  3  3  2  2  3  1 ND ND 21 0  17  4  6  19  5  6  2 ND ND 22 0 24  3  6  3  4  4  7 ND ND

[0074] The reference serum used is from a rabbit immunized with porcinezona pellucida using a primary injection with Freund's complete adjuvantand 2 booster injections with Freund's incomplete adjuvant. TABLE 3BEffect of adjuvants on the fertility of rabbits immunized againstporcine SIZP Live births/mating¹ Average live % reduction in ID No 1 2 3births/mating fertility Placebo  2  6  0  4 5.1 0 13  7  1 10 14  6  7 5 FCA  3  0  0  0 1.1 79 15  0  8  5 16  0 NM  0 MF 59  1 11 NM 10 6.10  7  0  0 NM 19  5 11 11 20  6  0  7 Alum 11  0  0  6 1.3 74 12  0  0 0 23  0  0  0 24  3  5  2 Gerbu without liposome encapsulation  9  0  011 5.3 0 10  0  0  0 21  8  9 11 22 10  7 11

Example 4 Immunocontraception in Cats

[0075] Twenty-nine specific pathogen free domestic short hair cats arehoused at the specific pathogen free facility at the University ofFlorida under the supervision of Dr. Julie Levy and Mr. Shawn Gorman.Estrus cycling is monitored by daily observation and vaginal cytology.Vaccine compositions of the present invention, placebo vaccines andserum samples are coded as part of a double-blind study. The cats aredivided randomly into 3 groups of nine or ten cats each. One groupreceives a placebo vaccine that contains all components of the vaccinecomposition except the antigen (porcine SIZP) by intramuscularinjection. Each cat in this group receives liposomes containing noantigen in saline (0.25 ml) suspended in FCA (0.25 ml). Each cat in asecond group of nine cats is immunized by intramuscular injection withthe vaccine composition containing SIZP (135 μg) encapsulated inliposomes in saline (0.25 ml) and suspended in FCA (0.25 ml). Each catin a third group of nine cats is immunized by intramuscular injectionwith the vaccine composition containing porcine SIZP (200 μg) with alum(0.12 ml, Pierce Chemical Co., catalogue number 77161) encapsulated inliposomes in saline (0.12 ml) and suspended in a suitablepharmacological carrier. Production of anti-SIZP antibodies in cats ismeasured by ELISA using protein A/alkaline phosphatase (Brown, R. G., W.D. Bowen, J. D. Eddington, W. C. Kimmins, M. Mezei, J. L. Parsons, B.Pohajdak. (1997) Temporal trends in antibody production in captive greyseals, harp and hooded seals to a single administrationimmunocontraceptive vaccine. J. Reproductive Immunology 35:53-64).

[0076] A single administration of the vaccine composition using FCAproduces anti-SIZP antibody titers that reached maximal titers within 2months (Table 4). The average two months post-immunization titer is58±2% of the reference serum which decreased to 41±4% of the referenceserum at four months post-immunization when a proven male cat isintroduced to the colony. Cats that receive a single administration ofthe vaccine composition using alum as adjuvant produce anti-SIZPantibodies with an average titer of 67±2% of the reference serum twomonths post-immunization.

[0077] Monthly serum samples from cats that are immunized with theplacebo vaccine containing all components of the vaccine compositionexcept the antigen, have an average anti-SIZP titer of 0.6±0.2% of thereference serum during the post-immunization monitoring period.Therefore, it is apparent that cats that received the placebo vaccinewill produce kittens during the post-immunization period. TABLE 4Production of anti-SIZP antibodies by cats immunized with the vaccinecomposition of the invention Anti-SIZP titer (% of reference serum) CatID Post-immunization (months) No. 0 1 2 3 4 5 6 7 8 9 10 11 Placebo  3 0 0  0  0  1  0  0  0  0  7  4  2  4 0  0  0  0  2  0  0  3  1  2  2  0 8 0  3  0  1  0  0  0  1  1  2  2  0 15 1  0  1  0  0  0  0  2  1 ND NDND A 0  0  3  0  1  0  0  0  0  2  2  0 B 0  0  0  1  1  1  0 ND  2  2 0  0 E 0  1  1  1  0  0  0  2  5 ND ND ND F 0  1  1  0  0  0  0  2  0ND ND ND I 0  1  0  1  1  0  0  0  2  0  0 ND N 0  1  0  0  1  1  1  2 1 ND ND ND Vaccine with FCA  1 0 60 57 68 46 56 23 46 15 40 23 23  2 060 53 58 44 25 23 69 46 68 70 62  9 0 47 56 41 56 20 58 78 103  74 86 ND13 0 42 64 53 66 55 47 29 33 12 16 ND 14 0 48 51 30 45 40 10 20  9 10 12 7 C 0 56 54 49 34 21 16 12 14 24 14 16 D 0 58 60 67 59 52 32 19 48 NDND ND L 0 47 61 40 28 16 46 37 59 70 59 72 G 0 59 65 79 85 81 92 94 96115  152  79 O 0 48 42 53 42 24 26 28 25 31 16 10 Vaccine with alum 1P 160 60 70 46 47 25 18 41 26  6 ND 1S 0 73 56 43 24 42 19 18 18 14  4 ND1T 0 62 62 58 30 34 14 12  8 11  4 ND 1V 2 70 68 60 40 36 12 12 ND ND NDND 1Y 1 84 82 84 81 80 67 34 44 28 ND ND 1Z 0 77 75 71 54 72 54 26 35 21 9 ND Z1 0 61 74 95 83 100  98 92 70 42 ND ND Z2 0 77 79 63 34 50 41 2818 11 ND ND Z3 0 65 72 61 55 30 35 18 20  6 ND ND Z4 0 73 ND 68 53 29 3217 12 13 ND ND

Example 5 Immunocontraception of Deer

[0078] Forty-one fallow deer (Dama dama) does on James Island, a360-hectare island that lies off the coast of southern British Columbia,are immunized with the vaccine composition using FCA as adjuvant.Another group of forty fallow deer does are immunized with the vaccinecomposition using alum as the adjuvant. For capture, the deer are baitedinto a large (200×200 meter) pen that is connected to a series of fencedenclosures and a raceway that terminates in a small building. Beforeimmunization, each deer is physically restrained and given a numberedear tag, a colored plastic collar or radio collar with a mortalitysensor, and a PIT (permanent identification transponder) tag bearing aunique code. Thus, if a treated deer loses all external marks, it couldstill be recognized as a treated animal from injury resulting from lossof ear tag and as a particular deer from the PIT tag. Each captive doeis injected intramuscularly in the rump with SIZP (100 μg) encapsulatedin liposomes with FCA or alum adjuvants. Untreated does serve ascontrols.

[0079] Anti-SIZP titers are measured as previously described (Brown, R.G., W. D. Bowen, J. D. Eddington, W. C. Kimmins, M. Mezei, J. L.Parsons, B. Pohjajdak. (1997) Temporal trends in antibody production incaptive grey seals, harp and hooded seals to a single administrationimmunocontraceptive vaccine. J. Reproductive Immunology 35:53-64) exceptthat protein G/alkaline phosphatase replaced protein A/alkalinephosphatase since protein G has a higher affinity for fallow deerimmunoglobulin than does protein A. Relative to the affinities ofprotein A and protein G for rabbit immunoglobulin (the reference serum),the affinities of protein A and protein G for fallow deer immunoglobulinare 8 and 89% respectively. Fallow deer anti-SIZP titers are uncorrectedfor relative affinity of protein G (Table 5A). None of the does examined2 months or more following the rut and 8-9 months after being immunizedwith the vaccine containing FCA were pregnant, while 96% (192/200)untreated does are pregnant. Pregnancy is determined by examination ofthe reproductive tract for signs of pregnancy or by analyzing blood fromlive captured does for pregnancy-specific protein B (PSPB) byBioTracking, Inc. of Moscow, Id. (Willard et al., “Pregnancy detectionand the effects of age, body weight, and previous reproductiveperformance on pregnancy status and weaning rates of farmed fallow deer(Dama dama). J. Animal Science. 77:32-38 (1999), the disclosure of whichis hereby incorporated by reference). The contrast between the pregnancyrates of immunized and unimmunized does shows clearly that the vaccinecomposition containing FCA is effective in preventing conception. Sincethis is a multiple year study, as many as possible of the does are livecaptured. TABLE 5A Production of anti-SIZP antibodies by fallow deerimmunized with the vaccine composition of the invention containing FCAadjuvant. Post-immunization anti-SIZP titer (% of reference serum)Fallow deer ID Time (months) No. 0 1-2 7-10 Controls 99023 0 0 0 99024 00 0 99025 0 0 0 99027 0 0 0 99028 0 0 0 99029 0 0 0 Vaccine with FCA99026 0 117 ND 99025 0 72 ND 2000-06 0 96 ND 2000-08 0 94 ND 99007 0 ND36 99008 0 ND 60 99009 0 ND 94 99016 0 ND 60 99017 0 ND 66 99019 0 ND133 99020 0 ND 56

[0080] Other experiments were performed on white-tailed deer. None ofthe white-tailed deer immunized with the vaccine composition comprisingFCA became pregnant one year post-immunization. Only one of thewhite-tailed deer immunized with the vaccine composition comprising alumdid not become pregnant one year post-immunization. The results foranti-SIZP titer levels are shown in Table 5B. TABLE 5B Production ofanti-SIZP antibodies by white-tailed deer immunized with a compositionof the present invention. White- tailed Anti-SIZP (% of reference serum)deer ID Post-immunization (months) No. 0 2 4 5 8 12 Freund's completeadjuvant (FCA)  19 0 ND ND  85 ND  21 0 ND ND 139 ND  33 0 ND ND 125 NDAlum 949 1 12 11 ND 48 27 916 1  6  4 ND  5  2 744 3 105  111  ND 123 75 694 0  7  7 ND  5  4 956 0  5  4 ND  2  2  9 0 ND ND  3 ND ND  14 0ND ND  8 ND ND  17 0 ND ND  4 ND ND  27 0 ND ND  4 ND ND

Example 6 The effect of Oil Content on the Production of Anti-SIZPAntibodies

[0081] The vaccine composition yields good antibody titers following asingle administration of an antigen, therefore, unless stated otherwiseall titers reported in the following example results from a singleadministration of the antigen in the vaccine formulation and otherimmunization protocols.

[0082] To determine if an aqueous phase is a necessary component of thevaccine composition to obtain a good immune response, three groups ofrabbits (2 or 3 rabbits/group) are immunized with three differentpreparations of the vaccine containing SIZP (50 μg SIZP/rabbit)encapsulated in liposomes that are suspended in saline (0.5 ml) andemulsified in Freud's complete adjuvant (0.5 ml). The proportion of oilphase and water phase is equal in these preparations (Table (6A). TABLE6A Effect of oil content of the vaccine composition on the production ofanti-SIZP antibodies by rabbits Anti-SIZP titer (% of reference serum)¹Oil content Post-immunization (months) (%, v/v) 0 1 2 3 4  50² 0 120 131  103  24 0 38 38 24 17  50² 0 91 91 54 67 0 46 112  143  112   50² 054 65 51 ND 0 95 94 81 ND 0 47 49 32 ND 100³ 0 61 75 136  34 0 14 24100  95 100⁴ 0 159  149  215  27 0 50 196  244  128  100⁴ 0 11 30 48 290 15 28 40 41 100⁴ 0 54 54 90 91 0 14  2 19 19 0 47 49 67 76

[0083] The vaccine formulated to contain no water is used to immunizefour groups of rabbits (2 or 3 rabbits/group) with four differentpreparations of the vaccine containing SIZP (50 μg SIZP/rabbit)encapsulated in freeze-dried liposomes suspended in Freund's completeadjuvant (0.2 ml or 0.5 ml). Since Freund's complete adjuvant containsno water, these preparations are water-free and contained only an oilphase. Average anti-SIZP titers 4 months post-immunization are 55±44%(coefficient of variation, cv 80%) for rabbits that are immunized withthe composition containing 50% oil and 59±41% (cv 69%) for rabbits thatare immunized with the vaccine containing 100% oil. There is nodifference in response of female rabbits that received the vaccine with100% oil and female rabbits that are immunized with the vaccinecontaining 50% oil (P=0.87; F (1,45) =0.03; average titers were 71±7%for 50% oil and 72±12% for 100% oil). These results indicate that thepresence of an aqueous phase is not necessary for a good immune responseto the vaccine.

[0084] To determine if there is a difference in duration of anti-SIZPtiters in rabbits that are immunized with the vaccine composition with50% and 100% oil, anti-SIZP titers are measured for 12 months (Table6B). Anti-SIZP titers during the 12 month post-immunization period aresimilar in rabbits immunized with 50% oil formulation and the rabbitimmunized with 100% oil formulation. To verify the biological effect ofimmunization with SIZP, proven female rabbits immunized with bothformulations of the vaccine are mated with proven males 3 times duringthe 12 month post-immunization period. Reduction in fertility was 80%for the rabbits that are immunized with the vaccine containing 50% oiland the female rabbit that is immunized with the vaccine containing 100%oil produce no offspring indicating that the biological effect ofreduced fertility is similar with both formulations of the vaccine.TABLE 6B Effect of oil content of the vaccine composition on theduration of anti-SIZP antibodies in rabbits Anti-SIZP titer (% ofreference serum) Rabbit Post-immunization (months) ID No. 0 1 2 3 4 5 67 11 12  50% oil content  3 0 145 112 94 82 45 23 24 20 20 15 0 100  7168 83 19 29 26 18 25 16 0 125 111 122  128  111  74 92 75 63 100% oilcontent  1 0 127 138 ND ND ND 33 51 17 27

[0085] Since liposomes are composed of material that is lipophilic,storage of liposomes in oil may lead to their destruction by dissolvingthe constituents of liposomes in the oil. To investigate this question,rabbits (2 rabbits in each group) are immunized with the vaccine (100%oil formulation) that is stored for up to 5 months at 5° C. and -20° C.Storage of the vaccine at 5° C. for 5 months reduced anti-SIZP titers ofrabbits by only 28% (Table 6C; P=0.002; F (5,33)=4.9). Storage of thevaccine at −20° C. for 5 months reduced anti-SIZP titers by only 14%(Table 6D; P=<0.001; F (5,35)=23.7). These results indicate that mostliposomes remain intact in oil since immunization of rabbits with asingle injection of SIZP suspended in Freund's complete adjuvant withoutliposome encapsulation results in low titers (Table 6E). TABLE 6C Effectof storage of the vaccine composition with 100% oil formulation on theproduction of anti-SIZP antibodies by rabbits Anti-SIZP titer¹ (% ofreference serum) Storage² Post-immmunization (months) (months) 0 1 2 3 45 6 Average SE 0 0 60 123 112 163 109 119 114 11.2 0 26 108 112 163 131141 1 0 26 112 183 121 122 100 113 11.9 0 77 133 142 117  70 153 2 0 63176 128  70 127  57  98 13.9 0 50 110 100 ND ND ND 3 0 23 138 168 104117 143  94 13.3 0 35 100 113  76  63  42 4 0 17  39 104  63  73 100  6810.1 0 47 136  73  26  45  85 5 0 22 127  69  66 140  99  82 11.8 0 27111  57  54 140  74

[0086] TABLE 6D Effect of storage of the vaccine composition with 100%oil formulation on the production of anti-SIZP antibodies by rabbitsAnti-SIZP titer¹ (% of reference serum) Storage² Post-immunization(months) (months) 0 1 2 3 4 5 6 Average SE 0 0 60 123 112 163 109 119114 11.2 1 26 108 112 163 131 141 1 0  9  13  13  41  19  60  27 6.7 0 6  16  14  73  33 ND 2 0  9  14  23  70 136 116  51 12.7 0  6  30  19 41  95  57 3 0 13  23 127 100  73  88  57 11.1 0 13  18  90  61  58  304 0 13  68  50  71  80 122  85 14.0 0 13 114 134  81 100 179 5 0  9 114 75  87 146  92  98 13.7 1 22 108 116 109 179 125

[0087] TABLE 6E Production of anti-SIZP antibodies by rabbits immunizedwith a single administration of SIZP without encapsulation of SIZP inliposomes Anti-SIZP titer (% of reference serum)¹ Rabbit IDPost-immunization (months) No. 0 1 2 3 4 1 0 43 19 9 2 2 0 27  7 4 1

[0088] To determine if the vaccine formulated to contain no aqueousphase would result in a good response in another mammalian species, greyseals (Halichoerus grypus) are immunized with the vaccine containingeither equal oil and aqueous phases, only an aqueous phase, or only anoil phase (Table 6F). There was no difference in anti-SIZP titers in thevaccine that contained equal oil and aqueous phases or only oil butadministration of the vaccine that contained all ingredients except oil,resulted in significantly lower titers. TABLE 6F Effect of oil contentof the vaccine composition on the production of anti-SIZP antibodies bygrey seals. Oil Anti-SIZP titer (% of reference serum)¹ contentPost-immunization (months) (%, v/v) 0 1 2 3 4  0² 0 5 4 1 1 0 1 2 1 1 50³ 0 7 9 145 107 0 41 52 82 38 100⁴ 0 20 28 79 60 0 3 30 90 50

Example 7 Use of Archaebacterial Lipids in Liposomes

[0089] Liposomes are completely closed lipid membranes that can be madefrom a variety of lipid materials. In this example, liposomes made usingarchaebacterial lipids are compared to liposomes made using soybeanlecithin for their ability to stimulate antibody production by rabbits(Table 7). Liposomes made with soybean lecithin result in betterproduction of anti-SIZP antibodies than liposomes made witharchaebacterial lipids. TABLE 7 Production of anti-SIZP antibodies byrabbits immunized with liposomes prepared with archaebacterial lipids orsoybean lecithin. Anti-SIZP titer (% of reference serum) Postimmunization (months) ID No. Type of lipid 0 1 2 3 4 5 112 Soybeanlecithin 0 143 195 137 197  98 115 Soybean lecithin 0 200 198 157 179106 117 Archaebacterial lipids 0 46 30 37 13 ND 118 Archaebacteriallipids 0 7 4 8 2 ND

Example 8 Immunization Against Streptokinase

[0090] The vaccine composition is unique in producing high titers ofanti-SIZP antibodies that are long-lasting following a singleadministration. To determine if the vaccine composition would producehigh antibody titers with other antigens, rabbits are immunized withstreptokinase.

[0091] Streptokinase is an exoprotein produced by pathogenic strains ofthe Streptococci family of bacteria. As an activator of vascularfibrinolysis its therapeutic usefulness has been appreciated for manyyears in the treatment of myocardial infarction. Streptokinase unfoldsin a non-cooperative manner. Therefore, the protein can assume a numberof partially folded states that contain some regions that appear to benative and others that are unfolded. Three domains of differentstability exist that are independent of other regions of the protein(Teuten et al., 1993, Biochem. J. 290:313-319). Native streptokinasecontains immunodominant epitopes in the C-terminal region (Torrens etal., 1999, Immunology Letters 70:213-218). The C-terminal region isrelatively unstructured (Parrado et al., 1996, Protein Sci 5:693-704)therefore heat treatment cannot alter the structure since it wasunstructured before heat treatment. The thermal stability of domain C issignificantly increased by its isolation from the rest of the chain(Connejero-Lara et al., 1996, Protein Sci 5:2583-2591). Loss of theC-terminal region results in a less immunogenic protein but does exposeimmunogenic epitopes hidden in the native molecule. In our studies, theC-terminal region was present in native and heat-treated streptokinase,therefore, as the immunodominant region of the protein, it would,determine the response of the rabbits. If the C-terminal region retainedthe same epitopes following heat treatment as found in the native state,one would not expect to find a difference in binding ofanti-streptokinase antibodies to native and heat-treated streptokinase.These are exactly the observations found (Table 8). We have proposedthat delivery of denatured proteins using a vaccine composition of thepresent invention favours the production of antibodies directed againstnative epitopes. This is supported by the studies of alcoholdehydrogenase in Example 2. The results with streptokinase areconsistent with this proposal since heat treatment would not alter thestructure of the immunodominant region and the prediction follows thatthere would be no difference in the immune response of rabbits beingimmunized with native and heat treated streptokinase regardless of thedelivery system employed. These are precisely our observations (Table8). TABLE 8 Epitope mapping of rabbit anti-streptokinase sera fromrabbits immunized with native and heat-treated streptokinase (100° C.for 10 minutes in 5% mercaptoethanol) using conventional immunizationprotocols¹ or the method of the present invention^(2.) Titer (% ofreference serum)³ Native Heat-treated Immunization streptokinasestreptokinase Rabbit Post-immunization (months) ID Antigen Delivery 0 12 0 1 2 21 Native Invention 0 100 122 0 98 122 24 Native Invention 0 8298 0 53 108 25 Native Conventional 0 10 89 0 8 100 20 NativeConventional 0 9 94 0 3 92 23 Heat- Invention 0 10 34 0 15 31 treated 28Heat- Invention 0 25 99 0 24 94 treated 27 Heat- Conventional 0 9 107 07 94 treated 30 Heat- Conventional 0 10 100 0 8 94 treated

[0092] It is evident from the results that a single injection ofstreptokinase using a vaccine of the present invention producedanti-streptokinase titers similar to titers obtained by the conventionalprimary and booster injection protocols. Also, regardless of theimmunization protocol used, that is the present invention orconventional, the antibodies produced bound to native and heat-treatedstreptokinase equally well.

Example 9 Use of an Edible Vegetable Oil

[0093] A vaccine composition was formulated in accordance with thisinvention using Canola oil in place of mineral oil. The results areshown in Table 9. The results indicate that vaccines formulated withCanola oil produce anti-SIZP antibodies in rabbits, therefore, Canolaoil is useful. However, the titer levels are not as high as with mineraloil. TABLE 9 Effect of Canola oil on production of anti-SIZP antibodiesin rabbits Anti-SIZP titer (% of reference serum) Rabbit VaccinePost-immunization (months) ID formulation 0 1 2 3 4 5 6 7 4 Alum/mineraloil 0 111 123 105 124  95 125 157 14 0 231 132 205 178 279 258 251 9FCA/mineral oil¹ 0 162 264 310 ND ND ND ND 26 0 351 166 112 ND ND ND ND34 FCA/Canola oil² 0 27 24 37 ND ND ND ND 36 0 18 24 36 ND ND ND ND

Example 10 Immunization Against Hepatitis B

[0094] A hepatitis B vaccine was formulated in accordance with thepresent invention using 5 micrograms hepatitis B surface antigen(Recombivax HB™, a recombinant hepatitis B antigen) containing alumadjuvant encapsulated in liposomes containing soybean lecithin (0.05 g)and cholesterol (0.005 g) suspended in saline (0.25 ml) then emulsifiedin low viscosity mineral oil (0.225 ml) and mannide oleate (0.025 ml). Aconventional hepatitis B vaccine using 5 micrograms hepatitis B surfaceantigen (Recombivax HB™) containing alum adjuvant in a volume of 0.5 mlaqueous medium as recommended by the manufacturer was also administered.Eight rabbits were immunized with the vaccine prepared in accordancewith the present invention and eight rabbits were immunized with theconventional vaccine. Results are shown in Table 10.

[0095] It is evident from Table 10 that the vaccine prepared inaccordance with the present invention results in about 6 times moreantibody 1 month post-immunization than conventional delivery ofhepatitis B surface antigen. TABLE 10 Production of anti-HepB antibodiesby rabbits immunized with a commercial HepB vaccine or with a HepBvaccine formulated in accordance with the present invention Anti-HepBtiter (mlU/ml)¹ Post-immunization Rabbit (months) ID 0 1 Commercialvaccine 96 0 736 101 0 1237 97 0 488 100 0 1877 99 0 6251 103 0 8384 980 688 102 0 1568 Average 0 2654 Vaccine of the invention 93 0 32,341 950 3371 88 0 5717 81 0 23,808 83 0 9344 84 0 17,856 79 0 9344 85 0 21,675Average 0 15,432 # hepatitis B surface antigen (anti-HBs) distributed byDiaSorin Inc., Stillwater, MN, USA.

Example 11 Effect of Formulating Vaccines with Alum Adjuvant Inside andOutside of Liposomes

[0096] Vaccines were prepared as follows: Group SIZP antigen Alum Medium1 inside liposome inside liposome saline 2 inside liposome outsideliposome saline 3 inside liposome inside liposome oil 4 inside liposomeoutside liposome oil 5 control-no liposomes oil 6 control-no liposomessaline

[0097] Group 1-4 were prepared with 100 μg SIZP encapsulated inliposomes formed with 0.1 g soybean lecithin and 0.01 g cholesterol. Theliposomes in Groups 1 and 3 also contained 100 μm ImjectAlum™. In Groups2 and 4, 100 μl ImjectAlum™ was placed outside the liposomes. In Groups1 and 2, the liposomes were suspended in 0.25 ml saline and thissuspension emulsified in 0.225 ml low viscosity mineral oil and 0.025 mlmannide oleate. In Groups 3 and 4, the liposomes were freeze dried thensuspended in 0.225 ml low viscosity mineral oil and 0.025 ml mannideoleate and this suspension emulsified in 0.25 ml saline. In Group 5, 100μg SIZP and 100 μl ImjectAlum™ were freeze dried, then suspended in0.225 ml low viscosity mineral oil and 0.025 ml mannide oleate andemulsified in 0.25 ml saline. In Group 6, 100 μg SIZP and 100 μlImjectAlum™ were freeze dried, then suspended in 0.25 ml saline andemulsified in 0.225 ml low viscosity mineral oil and 0.025 ml mannideoleate. Rabbits were immunized with the six groups of vaccines and theresults are shown in Table 11. TABLE 11 Production of anti-SIZPantibodies by rabbits immunized with four formulations of a vaccine ofthe present invention containing alum adjuvant (Groups 1-4) and twocontrol formulations containing alum adjuvant (Groups 5-6) Anti-SIZPtiter (% Standard reference serum) Average titer Error ofPost-immunization Post-immunization average Rabbit months months titerGroup ID 0 1 2 3 0 1 2 3 1 2 3 1 49 2 107 176 183 0 135 203 236 22 28 371 76 0 134 105 124 1 71 0 70 249 331 1 82 0 182 236 268 1 78 0 182 249274 2 73 0 373 273 304 0 287 207 258 32 24 15 2 42 0 328 199 281 2 62 0259 194 244 2 77 0 182 131 235 2 74 0 295 238 225 3 63 0 363 135 113 0300 171 160 32 26 34 3 67 0 286 175 140 3 70 0 332 241 261 3 80 0 218131 125 4 48 0 383 210 113 0 200 138 108 49 20 12 4 64 0 129 109 121 461 0 125 98 63 4 60 0 148 140 115 4 66 0 215 134 130 5 45 0 21 133 120 026 96 123 8 29 33 5 65 0 6 26 42 5 69 0 49 110 121 5 72 0 12 36 89 5 500 40 176 242 6 68 0 28 31 22 0 16 22 18 4 3 1 6 75 0 9 16 16 6 46 0 1823 17 6 43 0 10 19 16

Example 12 Effect of Formulating Vaccines with Heat Killed Mycobacteriumtuberculosis Adjuvant Inside and Outside of Liposomes

[0098] Vaccines were prepared as follows: Heat-killed M. Group SIZPantigen tuberculosis Medium 1 inside liposome inside liposome saline 2inside liposome outside liposome saline 3 inside liposome insideliposome oil 4 inside liposome outside liposome oil

[0099] Groups 1-4 were prepared with 100 μg SIZP encapsulated inliposomes formed with 0.1 g soybean lecithin and 0.01 g cholesterol. Theliposomes in Groups 1 and 3 also contained 200 μg heat killed M.tuberculosis. In Groups 2 and 4, 200 μg heat killed M. tuberculosis wasplaced outside the liposomes. In Groups 1 and 2, the liposomes weresuspended in 0.2 ml saline and this suspension emulsified in 0.18 ml lowviscosity mineral oil and 0.02 ml mannide oleate. In Groups 3 and 4, theliposomes were freeze dried then suspended in 0.18 ml low viscositymineral oil and 0.02 ml mannide oleate and this suspension emulsified in0.2 ml saline. Rabbits were immunized with the four groups of vaccinesand the results are shown in Table 12. TABLE 12 Production of anti-SIZPantibodies by rabbits immunized with four formulations of a vaccine ofthe present invention containing heat killed M. tuberculosis Anti-SIZPtiter (% Standard reference serum) Average titer Error ofPost-immunization Post-immunization average Rabbit months months titerGroup ID 0 1 2 3 0 1 2 3 1 2 3 1 33 0 245 236 259 0 318 262 437 51 18126 1 29 0 390 288 614 2 32 0 544 515 633 0 576 532 638 22 12 3 2 22 0608 549 642 3  9 0 162 264 310 0 293 599 508 93 237 140 3 13 0 424 933707 4 16 0 454 276 532 0 403 221 322 36 39 149 4 26 0 351 166 112

Example 13 Immunization of Rabbits with Native and Denatured YeastAlcohol Dehydrogenase (ADH) Together with Alum Adjuvant

[0100] Vaccines of the present invention were formulated containing 100μg of native or denatured ADH together with 100 μl ImjectAlum™encapsulated in liposomes formed with 0.1 g soybean lecithin and 0.01 gcholesterol. The liposomes were suspended in 0.25 ml saline and thesuspension emulsified in 0.225 ml low viscosity mineral oil and 0.025 mlmannide oleate.

[0101] Conventional vaccines were formulated containing 100 μg of nativeor denatured ADH together with 100 μl ImjectAlum™ and suspended in 0.5ml saline.

[0102] Denatured ADH was prepared by heating ADH to 100° C. for 30minutes and treating with 10% mercaptoethanol for 30 minutes at roomtemperature to cleave disulfide bonds. Mercaptoethanol was removed bydialysis for 12 hours and denatured ADH was recovered by freeze drying.

[0103] Results comparing the vaccines of the present invention to theconventional vaccines are shown in Table 13. TABLE 13 Production ofanti-ADH antibodies by rabbits immunized with native and denatured ADHusing alum as adjuvant Anti-ADH titer (% reference serum)Post-immunization (months) Rabbit Delivery Native ADH Denatured ADH IDSystem 0 1 2 3 0 1 2 3 Native ADH 54 invention 0 24 42 66 0 8 4 4 57 0100 143 146 0 29 3 6 55 0 91 85 111 0 26 3 5 40 conventional 0 1 1 2 0 10 1 44 0 1 1 1 0 1 0 1 47 0 5 4 8 0 3 1 1 Denatured ADH 53 invention 0 12 5 0 1 1 2 58 0 1 1 2 0 1 0.5 1 52 0 1 4 15 0 1 0.5 1 51 conventional 02 4 4 0 1 0.5 2 59 0 1 1 4 0 1 0.5 0 56 0 2 2 2 0 1 0.5 0

[0104] The results show that delivery of native or denatured ADH using aformulation of the present invention results in an increased productionof anti-ADH antibodies compared to the production of anti-ADH antibodiesby rabbits immunized against native or denatured ADH using conventionalmethods. Furthermore, rabbits immunized with denatured ADH produced moreantibodies directed against native ADH when a formulation of the presentinvention is used rather than when denatured ADH is delivered byconventional means with no booster injections.

Example 14 Epitope Mapping

[0105] Epitope mapping experiments to demonstrate that vaccines of thepresent invention produce antibodies having different bindingspecificity for an antigen than achieved by conventional immunizationprotocols of primary and secondary booster injections. Fragments of theZP antigen were specifically used but it is expected that other antigenswill behave in a similar manner.

[0106] Conventional immunization of grey and harp seals with a primaryinjection and two booster injections results in low anti-SIZP antibodytiters that peak two months post-immunization in both grey and harpseals. In contrast, immunization with a vaccine formulated in accordancewith the present invention produces anti-SIZP antibody titers thatpersist for at least 24 months in grey seals and 5-6 months in harpseals, with one exception. Titers in harp seals reach a plateau thatpersist for 6-10 months post-immunization. Therefore, a vaccineformulated in accordance with the present invention induces highanti-SIZP titers with long duration compared to conventionalimmunization protocols using primary and booster injections.

[0107] The polypeptide fragments of ZPB and ZPC that were used inepitope mapping to demonstrate that anti-SIZP antibodies producedfollowing conventional immunization protocols have a different bindingspecificity than anti-SIZP antibodies produced following immunizationwith a vaccine of the present invention are shown in FIG. 1. Thefragments ZPB1, ZPB2, ZPC1 and ZPC2 are short length polypeptides and donot have the three-dimensional structures of full length ZPB and ZPC. InFIG. 1, the full-length unprocessed polypeptides are shown above the twoZPB and ZPC fragments. The secretory signal peptides that are cleaved inthe native proteins are shaded in black.

[0108] Anti-SIZP grey seal antibodies produced following conventionalimmunization (a primary and two booster injections using FCA adjuvant)have a high affinity for the ZPB1, ZPB2, ZPC1 and ZPC2 fragments (Table14A, seal ID 1). In contrast, grey seals immunized with a vaccineformulated in accordance with the present invention (Table 14A, seal ID76 and 96) produce antibodies that have a low affinity for fragmentsZPB2, ZPC1 and ZPC2 one year post-immunization and low affinity for allfour fragments three years post-immunization. The four fragmentstogether account for 80% of the protein found in SIZP. TABLE 14A Epitopemapping of grey seal anti-SIZP antibodies using recombinant fragments ofZPB and ZPC produced in E. coli Post- Seal immunization Binding relativeto SIZP (%) ID (months) ZPB1 ZPB2 ZPC1 ZPC2 Total 1 3 30 44 59 41 174 14 71 70 83 63 287 76 12 54 25 8 12 99 76 36 18 9 13 11 51 96 12 47 18 1510 90 96 36 10 8 15 10 43

[0109] A temporal study of the binding specificity of antibodiesproduced by grey seals immunized with a vaccine formulated in accordancewith the present invention indicated that antibodies produced earlypost-immunization (<7 months) bind to epitopes found predominantly onthe ZPB1 fragment. Antibodies produced late post-immunization (>7months) have lower affinity for ZPB1 and the other three fragments.ZPB1, ZPB2, ZPC1 and ZPC2 are low molecular weight and are notglycosylated. These fragments have less three-dimensional structure thanfull-length ZPB and ZPC because of their low molecular weight.Therefore, antibodies that bind to SIZP but not ZPB1, ZPB2, ZPC1 or ZPC2must either be recognizing three-dimensional structures found only onfull-length ZPB and ZPC or the carbohydrate covalently linked to theseproteins. Since the total amount of antibody bound to the fragmentsearly post-immunization exceeds or is equivalent to the amount ofantibody binding to ZPB and ZPC, carbohydrate-recognizing antibody musthave a minor role. This implies that 3-D structures determine thedifference in binding to the fragments as opposed to SIZP. A survey ofnine other grey seals immunized with SIZP in a vaccine of the presentinvention indicates similar reduction to antibodies produced 5 months ormore post-immunization.

[0110] In another experiment, three of four rabbits immunized with avaccine of the present invention produced antibodies with a higheraffinity for epitopes in ZPB1 than in the other three fragments. Only20-40% of the antibodies produced by all four rabbits bound to epitopesfound in the four ZP fragments. Therefore, 60-80% of the anti-SIZPantibodies produced by rabbits immunized with a vaccine of the presentinvention bound only to epitopes found in full length ZPB and ZPC.Therefore, 60-80% of antibodies produced in rabbits immunized with avaccine of the present invention recognize epitopes related to native3-D structures.

[0111] In yet another experiment, immunization of harp seals (156 and162) by conventional protocols of a primary injection using FCA adjuvantfollowed by booster injections with FIA adjuvant produced antibodiesearly post-immunization that bound to epitopes found in ZPB1, ZPB2 andZPC2 (harp seal 156) or all four fragments (harp seal 162) as well as inSIZP (Table 14B). In contrast, immunization of harp seal 151 with avaccine formulated in accordance with the present invention producedantibodies early post-immunization (<5 months) that bound to epitopesfound in all four ZP fragments but antibodies produced latepost-immunization (>7 months) bound to epitopes found only on fulllength ZPB and ZPC (Table 14B). Only 30-40% of the antibodies producedby immunization of harp seal 153 with a vaccine of the present inventionbound well to epitopes on the four ZP fragments, implying that 60-70% ofthe antibodies produced by harp seal 153 during the 7 monthpost-immunization period bound only to epitopes found in full length ZPBand ZPC. These epitopes must be related to structures found only in fulllength ZPB and ZPC implying 3-D structures. Immunization of hooded seal1 with a vaccine of the present invention produced antibodies with asimilar temporal sequence of specificity as harp seal 151. TABLE 14BEpitope mapping of harp and hooded seal anti-SIZP antibodies usingrecombinant fragments of ZPB and ZPC produced in E. coli Post-immunization Binding relative to SIZP (%) Seal ID (months) ZPB1 ZPB2ZPC1 ZPC2 Total Harp 156 2 9 8 7 7 31 3 30 19 10 24 83 4 36 38 2 34 110Harp 162 2 8 10 11 11 40 3 33 26 19 24 102 Harp 151 1 40 33 36 33 142 341 28 32 21 122 5 21 27 35 34 117 6 50 26 30 44 150 7 8 6 9 8 31 9 4 1732 10 63 Harp 153 2 12 6 11 9 38 3 16 12 9 12 49 4 6 5 7 3 21 5 13 8 1211 44 6 12 9 11 12 44 7 9 6 0 7 22 Hooded 1 2 30 22 26 27 105 3 33 25 2830 116 4 37 32 38 34 141 5 31 24 29 31 115 7 15 12 12 11 50 8 12 13 11 844

claims:
 1. A composition for use as a vaccine, comprising: (a) a carriercomprising a continuous phase of a hydrophobic substance; (b) liposomes;(c) an antigen; and, (d) a suitable adjuvant.
 2. The composition ofclaim 1, wherein the hydrophobic substance is a liquid.
 3. Thecomposition of claim 2, wherein the carrier is an oil or a water-in-oilemulsion.
 4. The composition of claim 3, wherein the oil is mineral oil,a vegetable oil or a nut oil.
 5. The composition of claim 3, wherein theadjuvant is alum, another compound of aluminum or TiterMax.
 6. Thecomposition of claim 5, wherein the adjuvant is alum.
 7. The compositionof claim 3, wherein the antigen is a suitable native, non-native,recombinant or denatured protein or peptide, or a fragment thereof. 8.The composition of claim 7, wherein the antigen is a viral, bacterial,protozoal or mammalian antigen.
 9. The composition of claim 8, whereinthe antigen is capable of eliciting an antibody that recognizes a nativeepitope.
 10. The composition of claim 9, wherein the native epitope isin a mammal.
 11. The composition of claim 10, wherein the mammal is ahorse, a rabbit, a deer or a cat.
 12. The composition of claim 7,wherein the antigen is zona pellucida, alcohol dehydrogenase, hepatitisB or streptokinase.
 13. The composition of claim 3, wherein theliposomes comprise unesterified cholesterol and a phospholipid with atleast one head group selected from the group consisting ofphosphoglycerol, phosphoethanolamine, phosphoserine, phosphocholine andphosphoinositol.
 14. The composition of claim 3, wherein the liposomescomprise lipids in phospholipon 90 G.
 15. The composition of claim 3which is essentially free of lipid A.
 16. The composition of claim 4,wherein the antigen is zona pellucida, the adjuvant is alum, and thevaccine provides effective long-term immunocontraception in a mammal.17. The composition of claim 16, wherein the oil is mineral oil and thecomposition is essentially free of lipid A.
 18. A method forpotentiating an immune response in an animal, which method comprisesadministering to the animal an effective amount of a vaccine compositioncomprising: (a) a carrier comprising a continuous phase of a hydrophobicsubstance; (b) liposomes; (c) an antigen; and, (d) a suitable adjuvant.19. The method of claim 18, wherein the hydrophobic substance is aliquid.
 20. The method of claim 18, wherein the carrier is an oil or awater-in-oil emulsion.
 21. The method of claim 20, wherein the oil ismineral oil, a vegetable oil or a nut oil.
 22. The method of claim 21,wherein the adjuvant is alum.
 23. The method of claim 21, wherein theantigen is zona pellucida, alcohol dehydrogenase, hepatitis B orstreptokinase.
 24. The method of claim 20, wherein the antigen iscapable of eliciting an antibody that recognizes a native epitope. 25.The method of claim 24, wherein the native epitope is in a mammal. 26.The method of claim 25, wherein the mammal is a horse, a rabbit, a deeror a cat.
 27. The method of claim 20, wherein the composition issubstantially free of lipid A.
 28. A method of preparing a vaccinecomposition comprising the steps of: (a) encapsulating an antigen or anantigen/adjuvant complex in liposomes to form liposome-encapsulatedantigen; (b) mixing the liposome-encapsulated antigen with a carriercomprising a continuous phase of a hydrophobic substance; and, (c)adding a suitable adjuvant if an antigen/adjuvant complex is not used inpart (a).
 29. The method of claim 28, wherein the liposome-encapsulatedantigen is freeze-dried.
 30. The method of claim 29, wherein an antigenwithout adjuvant is encapsulated in the liposomes before adding theadjuvant and the liposome-encapsulated antigen is freeze-dried afteradding the adjuvant to form a freeze-dried liposome-encapsulated antigenwith external adjuvant.
 31. The method of claim 30, wherein the adjuvantis added to pyrogen-free water before the adjuvant is added to theliposome-encapsulated antigen.
 32. The method of claim 31, wherein thefreeze-dried liposome-encapsulated antigen with external adjuvant ismixed with the carrier, and wherein an aqueous medium is mixed with thecarrier to form an emulsion of water-in-the hydrophobic substance. 33.The method of claim 30, wherein the freeze-dried liposome-encapsulatedantigen with external adjuvant is then mixed with the carrier.
 34. Themethod of claim 29, wherein the liposome-encapsulated antigen comprisesan antigen/adjuvant complex, and wherein the freeze-driedliposome-encapsulated antigen is mixed with the carrier, and wherein anaqueous medium is mixed with the carrier to form an emulsion ofwater-in-the hydrophobic substance.
 35. The method of claim 28, wherein:(i) the liposome-encapsulated antigen is mixed with an aqueous mediumbefore being mixed with the carrier; (ii) the adjuvant is added to thecarrier before the carrier is mixed with the liposome-encapsulatedantigen; and, (iii) the carrier is mixed with the liposome-encapsulatedantigen to form an emulsion of water-in-the hydrophobic substance. 36.The method of claim 28, wherein: (i) the liposome-encapsulated antigencomprises an antigen/adjuvant complex; (ii) the liposome-encapsulatedantigen is mixed with an aqueous medium before being mixed with thecarrier; and, (iii) the liposome-encapsulated antigen is mixed with thecarrier to form an emulsion of water-in-the hydrophobic substance. 37.The method of claim 28, wherein the hydrophobic substance is a liquid.38. The method of claim 37, wherein the liquid is an oil.
 39. The methodof claim 38, wherein the oil is mineral oil.
 40. The method of claim 28,wherein the adjuvant is alum.
 41. The method of claim 28, wherein theantigen is zona pellucida, alcohol dehydrogenase, hepatitis B orstreptokinase.
 42. The method of claim 28, wherein the adjuvant is alumand the carrier is an oil or a water-in-oil emulsion.
 43. The method ofclaim 33, wherein the oil is mineral oil.